miR155 deficiency enhanced the susceptibility to T. gondii
MiR-155 has recently been uncovered to be contributed to the resistance to the experimental parasitic diseases [16, 17]. To test if the lack of miR-155 would potentiate the chronic T. gondii in mice models, miR-155−/− mice were orally inoculated with 10 PRU tissue cysts. All of the miR-155−/− infected mice died after 17 days of infection, but WT mice infected with T. gondii showed a prolonged survival time (Fig. 1A). To further confirm the T. gondii infection in WT and miR-155−/− mice, the mean brain cyst loadings were counted. T. gondii-infected miR-155−/− mice showed more brain cyst (Fig. 1B). These data demonstrate that the absence of miR-155 enhance susceptibility to T. gondii in mice models.
miR155 deficiency impairs both Th1 and Th2 immune responses in the spleens of T. gondii-infected mice.
As miR-155 has been shown to be critical for regulating T cell responses as well as inflammatory responses and cytokine signals, which have the well-documented roles in the control of intracellular pathogens, including T. cruzi and Leishmania donovani [16, 17], we further analyzed the production of cytokines Th1-associated IFN-γ, IL-2, Th2-associated IL-4 and IL-13, Th17-associated IL-17A and IL-17F by splenic cells harvested from WT and miR155KO (miR-155−/−) mice after infection with T. gonii PRU cysts.
After ELISA analysis of TLA-stimulated spleen cells supernatants, it has been shown that the production of IFN-γ and IL-2 was significantly reduced in T. gondii-infected miR-155−/− mice compared to WT controls (Fig. 2). Likewise, the IL-13 production by splenic cells from T. gondii-infected miR-155−/− mice were dramatically decreased in contrast to WT controls (Fig. 2). However, there were no significant changes of the levels of IL-4, and IL-17A and IL-17F in these T. gondii-infected miR-155−/− mice and WT controls (Fig. 2). Taken together, these data suggest that this T. gondii infection in miR-155−/− is due to the lower protective cytokines production.
Lack of miR155 contributes to the impaired recruitments of CD4+ T, CD8+ T, NK and NK-T cells in the spleens of T. gondii-infected mice.
NK cells, NK-T, and T cells are usually recruited to kill T. gondii by hosts through the production of IFN-γ and the perforin-independent mechanism [10, 22]. To investigate whether the miR155 deficiency has an effect in these immune cells recruitment, the splenic cell population of CD4+ T cells, CD8+ T cells, NK cells and NK-T cells were analyzed from T. gondii-infected miR155−/− and WT mice by flow cytometry. We have found that these indicated splenic cell populations of T. gondii-infected miR-155−/− mice have been significantly decreased in contrast to WT counterparts (Fig. 3). These results suggest that miR155 plays a role in mediating recruitment of NK, NK-T cells, CD4+ T cells and CD8+ T cells, that control the T. gondii infection, while these decreased recruited cells in miR-155−/− mice could not activate the protective immunity effectively, leading to the subsequent aggravated parasitic infection and even death.
miR155 deficiency exacerbated CD8+ T cell exhaustion
Since miR-155 have recently demonstrated to restrain CD8+ T cell functional exhaustion in chronic virus infection and tumor, through targeting to several inhibitors of cytokine signaling, including SOCS1, SHIP1 [23, 24]. Thus, to better understand the role of miR-155 on CD8+ T cell exhaustion during chronic infection with T. gondii, we analyzed the expression of IFN-γ, TFN-α and PD-1 in CD8+ T cells and the number of regulatory cells (Tregs) by flow cytometry (Fig. 4). As we expected, miR155KO mice significantly decreased the numbers of IFN-γ CD8+ and TNF-α CD8+ T cells in contrast to WT mice, suggesting a decreased effector T cells in miR155KO mice (Fig. 4). In the meanwhile, the number of Tregs were also up-regulated by T. gondii-infected miR155KO mice, suggesting the enhanced immune-inhibitory effects followed by the deficient expression of miR155.
It is known that PD-1 expression by the myeloid cell population plays an important role in immune regulation in various infectious diseases as well as in several cancer models [25–27]. Since PD-1 has been implicated in suppressing T cell response in T. gondii , flow cytometric analysis of the expression PD-1 in CD8+ T cell revealed that T. gondii-infected miR155KO mice up-regulated the expression PD-1 in CD8+ T cell in contrast to T. gondii-infected WT mice. Furthermore, miR155KO mice showed increased expression of both SHIP-1 and SOCS1 by qRT-PCR (Fig. 4), which are known to be direct targets of miR155 [23, 24]. Together, these findings indicate that miR155 deficiency contribute to the increased T cell exhaustion by suppressing T cell responses via targeting to SHIP-1 and SOCS1.
miR155 deficiency decreased accumulation of splenic inflammatory monocytes and DCs and expression of pro-inflammatory mediators.
Recent studies have established the critical role of CD11b+Ly6C+ cells and DCs in T. gondii infection [29, 30]. Flow cytometric analysis of phagocyte populations has revealed that T. gondii-infected miR-155−/− mice contained significantly lower numbers of CD11b+Ly6C+ cells and DCs than their WT counterparts (Fig. 5). In the meanwhile, we further determined whether decreased numbers of CD11b+Ly6C+ cells and DCs in miR155KO mice consequently impaired the expression of pro-inflammatory mediators by Flow cytometric analysis. In consistent with decreased numbers of CD11b+Ly6C+ cells and DCs, the expression of IL-1α, IL-1β and IL-6 was significantly reduced in contrast to their T. gondii-infected WT counterparts (Fig. 5). These data suggest that miR155 deficiency leads to the decreased accumulation of DCs and Ly6C+ inflammatory monocytes, in combination with reduced production of pro-inflammatory mediators could contribute to high parasitic burdens in miR155KO mice.